Evaluation of novel epibatidine analogs in the rat nicotine drug discrimination assay and in the rat chronic constriction injury neuropathic pain model.

Nicotine is the primary psychoactive component responsible for maintaining tobacco dependence in humans. Chronic pain is often a consequence of tobacco-related pathologies, and the development of a dual therapeutic that could treat chronic pain and tobacco dependence would be advantageous. Epibatidine reliably substitutes for nicotine in the drug discrimination assay, and is a potent analgesic, but has a side-effect profile that limits its therapeutic potential. Thus, considerable efforts to produce epibatidine derivatives are underway. Here we tested three epibatidine derivatives, 2'-fluoro-3'-(4-nitrophenyl)deschloroepibatidine (RTI-7527-102; i.e., RTI-102), 2'-fluorodeschloroepibatidine (RTI-7527-36; i.e., RTI-36), and 3'-(3″-dimethylaminophenyl)-epibatidine (RTI-7527-76; i.e., RTI-76) in both the rat nicotine drug discrimination assay as well as in the rat chronic constriction injury (CCI) of the sciatic nerve neuropathic pain model. Male and female Sprague-Dawley rats were trained on a fixed-ratio 10 schedule to discriminate nicotine (0.32 mg/kg base) from vehicle. All compounds dose-dependently substituted for nicotine, without significant decreases in response rates. In the discrimination assay the rank order potency was RTI-36 > nicotine > RTI-102 > RTI-76. Evidence suggests the α4ß2* subtype is particularly important to nicotine-related abuse potential. Thus, here we utilized the antagonist dihydro-ß-erythroidine (DHßE) to examine relative ß2 subunit contribution. DHßE (3.2 mg/kg, s.c.) antagonized the discriminative stimulus effects of nicotine. However, relative to antagonism of nicotine, DHßE produced less antagonism of RTI-102 and RTI-76 and greater antagonism of RTI-36. It is likely that at nicotinic receptor subunits RTI-102, RTI-76 and RTI-36 possess differing activity. To confirm that the full discriminative stimulus of these compounds was due to nAChR activity beyond the ß2 subunit, we examined these compounds in the presence of the non-selective nicotinic receptor antagonist mecamylamine. Mecamylamine (0.56 mg/kg, s.c.) pretreatment abolished nicotine-paired lever responding for all compounds. In a separate cohort, male and female Sprague-Dawley rats underwent CCI surgery and tested for CCI-induced mechanical allodynia via the von Frey assay. Each compound produced CCI-induced mechanical allodynia reversal. RTI-36 displayed higher potency than either RTI-102 or RTI-76. These novel epibatidine analogs may prove to be useful tools in the fight against nicotine dependence as well as novel neuropathic pain analgesics.

Evaluation of the terpenes ß-caryophyllene, α-terpineol, and γ-terpinene in the mouse chronic constriction injury model of neuropathic pain: possible cannabinoid receptor involvement.

Pain is one of the most common reasons to seek medical attention, and chronic pain is a worldwide epidemic. Anecdotal reports suggest cannabis may be an effective analgesic. As cannabis contains the terpenes α-terpineol, ß-caryophyllene, and γ-terpinene, we hypothesized these terpenes would produce analgesia in a mouse model of neuropathic pain. We used the chronic constriction injury of the sciatic nerve mouse model, which produces mechanical allodynia, assessed via the von Frey assay, as well as thermal hyperalgesia assessed via the hotplate assay. Compounds were further assessed in tests of locomotor activity, hypothermia, and acute antinociception. Each terpene produced dose-related reversal of mechanical allodynia and thermal hyperalgesia. Thermal hyperalgesia displayed higher sensitivity to the effects of each terpene than mechanical allodynia, and the rank order potency of the terpenes was α-terpineol > ß-caryophyllene > γ-terpinene. To examine the involvement of cannabinoid receptors, further tests were conducted in mice lacking either functional cannabinoid type 1 receptors (CB1R (-/-)) or cannabinoid type 2 receptors (CB2R (-/-)). Compared to wild type mice, CB1R (-/-) mice treated with α-terpineol displayed a 2.91-fold decrease in potency to reverse mechanical allodynia; in CB2R (-/-) mice, the potency of α-terpineol was decreased 11.73-fold. The potency of ß-caryophyllene to reverse mechanical allodynia decreased 1.80-fold in CB2R (-/-) mice. Each terpene produced a subset of effects in tests of locomotor activity, hypothermia, and acute antinociception. These findings suggest α-terpineol, ß-caryophyllene, and γ-terpinene may have differential cannabinoid receptor activity and a pharmacological profile that may yield new efficacious analgesics.

Slow conformational dynamics of the human A2A adenosine receptor are temporally ordered.

A more complete depiction of protein energy landscapes includes the identification of different function-related conformational states and the determination of the pathways connecting them. We used total internal reflection fluorescence (TIRF) imaging to investigate the conformational dynamics of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR), at the single-molecule level. Slow, reversible conformational exchange was observed among three different fluorescence emission states populated for agonist-bound A2AAR. Transitions among these states predominantly occurred in a specific order, and exchange between inactive and active-like conformations proceeded through an intermediate state. Models derived from molecular dynamics simulations with available A2AAR structures rationalized the relative fluorescence emission intensities for the highest and lowest emission states but not the transition state. This suggests that the functionally critical intermediate state required to achieve activation is not currently visualized among available A2AAR structures.

Alterations in mouse spinal cord and sciatic nerve microRNAs after the chronic constriction injury (CCI) model of neuropathic pain.

Pain is one of the most common reasons to seek medical attention and chronic pain is a worldwide epidemic. There are currently no relevant biomarkers for the diagnosis of chronic pain, and new therapeutic strategies for chronic pain treatment are desperately needed. The chronic constriction injury (CCI) of the sciatic nerve is a widely used preclinical model of pathological neuropathic pain. Over the past decade, investigators have come to appreciate the many contributions of noncoding RNA including microRNA (miRNA), and other long and short noncoding (nc) RNAs. The development and/or maintenance of chronic pain could be controlled epigenetically through ncRNAs. Here we seek to characterize CNS tissues in a mouse model of neuropathic pain as this may serve to elucidate potential biomarkers relevant to pathological pain in humans. Male C57BL6/J mice (6 CCI and 6 sham procedure) underwent surgery for sciatic nerve ligation with chromic gut sutures. Following 7 days, mechanical allodynia was quantified using the von Frey assay. Mice were then euthanized for collection of spinal cord and sciatic nerve. cDNA was synthesized to 627 unique mature miRNAs from the total RNA. In the CCI mice that displayed mechanical allodynia, 11 and 125 miRNAs were differentially expressed (i.e., greater than 1.5-fold increase or decrease; P < 0.05) in the spinal cord and sciatic nerve, respectively, as compared to sham controls. Among those differentially expressed miRNAs in the sciatic nerve of CCI mice, the following passed the more stringent Bonfferoni correction: miR-138-3p, miR-138-5p and miR-676-3p, reduced and miR-142-5p, increased. Our data support miRNAs as promising therapeutic targets for the treatment of pathological pain.

The discriminative stimulus effects of epibatidine in C57BL/6J mice.

The α4ß2* nicotinic acetylcholine receptor (nAChR) subtypes are targeted for the development of smoking cessation aids, and the use of drug discrimination in mice provides a robust screening tool for the identification of drugs acting through nAChRs. Here, we established that the α4ß2* nAChR agonist epibatidine can function as a discriminative stimulus in mice. Male C57BL/6J mice discriminated epibatidine (0.0032 mg/kg, subcutaneously) and were tested with agonists varying in selectivity and efficacy for α4ß2* nAChRs. The discriminative stimulus effects of epibatidine were characterized with the nonselective, noncompetitive nicotinic antagonist mecamylamine, with the selective ß2-substype-containing nAChR antagonist dihydro-ß-erythroidine hydrobromide (DHßE), and the α7 antagonist methyllycaconitine (MLA). Nicotine (0.32-1.0 mg/kg, subcutaneously), the partial nAChR agonist cytisine (1.0-5.6 mg/kg, subcutaneously), and the α7 nAChR agonist N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide (10-56 mg/kg, intraperitoneally) produced no more than 33% epibatidine-appropriate responding. The partial α4ß2* nAChR agonists varenicline and 2'-fluoro-3'-(4-nitro-phenyl)deschloroepibatidine produced 61 and 69% epibatidine-appropriate responding, respectively. DHßE and mecamylamine, but not MLA, significantly antagonized the discriminative stimulus effects of epibatidine. These results show that epibatidine may be trained as a discriminative stimulus in mice and has utility in elucidating the in-vivo pharmacology of α4ß2* nAChR ligands.

Advances in the In vitro and In vivo pharmacology of Alpha4beta2 nicotinic receptor positive allosteric modulators.

Receptors containing α4 and ß2 subunits are a major neuronal nicotinic acetylcholine receptor (nAChR) subtype in the brain. This receptor plays a critical role in nicotine addiction, with potential smoking cessation therapeutics producing modulation of α4ß2 nAChR. In addition, compounds that act as agonists at α4ß2 nAChR may be useful for the treatment of pathological pain. Further, as the α4ß2 nAChR has been implicated in cognition, therapeutics that act as α4ß2 nAChR agonists are also being examined as treatments for cognitive disorders and neurological diseases that impact cognitive function, such as Alzheimer's disease and schizophrenia. This review will cover the molecular in vitro evidence that allosteric modulators of the α4ß2 neuronal nAChR provide several advantages over traditional α4ß2 nAChR orthosteric ligands. Specifically, we explore the concept that nAChR allosteric modulators allow for greater pharmacological selectivity, while minimizing potential deleterious off-target effects. Further, here we discuss the development and preclinical in vivo behavioral assessment of allosteric modulators at the α4ß2 neuronal nAChR as therapeutics for smoking cessation, pathological pain, as well as cognitive disorders and neurological diseases that impact cognitive function. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology'.

Unexpected loss of sensitivity to the nicotinic acetylcholine receptor antagonist activity of mecamylamine and dihydro-ß-erythroidine in nicotine-tolerant mice.

OBJECTIVES: There is a long-standing interest in developing nicotinic acetylcholine receptor (nAChR) antagonists for concomitant use with nAChR agonists (e.g., nicotine replacement) as complementary smoking cessation aids. Previous studies demonstrate that daily nicotine treatment confers tolerance to some effects of nicotine, as well as cross-tolerance to other nAChR agonists. The current study assessed the extent to which antagonism of nicotine varies as a function of daily nicotine treatment. METHODS: Schedule-controlled responding and hypothermia were selected for study because they have been previously used to examine the pharmacology of nicotine, and both are sensitive to the development nicotine tolerance. The rate-decreasing and hypothermic effects of nicotine, as well as antagonism of those effects, were examined in C57BL/6J mice before, during treatment with, and after discontinuation of three daily injections of 1.78 mg/kg nicotine. The nonselective nAChR antagonist mecamylamine and the ß2 nAChR antagonist dihydro-ß-erythroidine (DHßE) were studied in combination with nicotine. RESULTS: The ED50 values of nicotine to produce rate-decreasing and hypothermic effects were, respectively, 0.44 and 0.82 mg/kg prior, 1.6 and 3.2 mg/kg during, and 0.74 and 1.1 mg/kg after discontinuation of daily nicotine treatment. Prior to daily nicotine treatment, mecamylamine decreased response rate and rectal temperature. However, during daily nicotine, mecamylamine (up to 5.6 mg/kg) only decreased rectal temperature. DHßE (up to 5.6 mg/kg) when studied prior to daily nicotine decreased rectal temperature, but that decrease was abolished during chronic nicotine treatment. Mecamylamine and DHßE antagonized the rate-decreasing and hypothermic effects of nicotine before and after daily nicotine; however, during daily nicotine, mecamylamine and DHßE antagonized only the hypothermic effects of nicotine. CONCLUSIONS: The differential antagonism of rate-decreasing and hypothermic effects implicates differential involvement of nAChR subtypes. The decreased capacity of mecamylamine and DHßE to antagonize nicotine during chronic nicotine treatment may indicate that their effectiveness as smoking cessations might vary as a function of nicotine tolerance and dependence.

Investigation of the Adrenergic and Opioid Binding Affinities, Metabolic Stability, Plasma Protein Binding Properties, and Functional Effects of Selected Indole-Based Kratom Alkaloids.

Selected indole-based kratom alkaloids were evaluated for their opioid and adrenergic receptor binding and functional effects, in vivo antinociceptive effects, plasma protein binding, and metabolic stability. Mitragynine, the major alkaloid in Mitragyna speciosa (kratom), had higher affinity at opioid receptors than at adrenergic receptors while the vice versa was observed for corynantheidine. The observed polypharmacology of kratom alkaloids may support its utilization to treat opioid use disorder and withdrawal.

Discriminative stimulus effects of mecamylamine and nicotine in rhesus monkeys: Central and peripheral mechanisms.

Mecamylamine is a non-competitive nicotinic acetylcholine receptor (nAChR) antagonist that has been prescribed for hypertension and as an off-label smoking cessation aid. Here, we examined pharmacological mechanisms underlying the interoceptive effects (i.e., discriminative stimulus effects) of mecamylamine (5.6 mg/kg s.c.) and compared the effects of nAChR antagonists in this discrimination assay to their capacity to block a nicotine discriminative stimulus (1.78 mg/kg s.c.) in rhesus monkeys. Central (pempidine) and peripherally restricted nAChR antagonists (pentolinium and chlorisondamine) dose-dependently substituted for the mecamylamine discriminative stimulus in the following rank order potency (pentolinium > pempidine > chlorisondamine > mecamylamine). In contrast, at equi-effective doses based on substitution for mecamylamine, only mecamylamine antagonized the discriminative stimulus effects of nicotine, i.e., pentolinium, chlorisondamine, and pempidine did not. NMDA receptor antagonists produced dose-dependent substitution for mecamylamine with the following rank order potency (MK-801 > phencyclidine > ketamine). In contrast, behaviorally active doses of smoking cessation aids including nAChR agonists (nicotine, varenicline, and cytisine), the smoking cessation aid and antidepressant bupropion, and the benzodiazepine midazolam did not substitute for the discriminative stimulus effects of mecamylamine. These data suggest that peripheral nAChRs and NMDA receptors may contribute to the interoceptive stimulus effects produced by mecamylamine. Based on the current results, the therapeutic use of mecamylamine (i.e., for smoking or to alleviate green tobacco sickness) should be weighed against the potential for mecamylamine to produce interoceptive effects that overlap with another class of abused drugs (i.e., NMDA receptor agonists).

Nicotine-like discriminative stimulus effects of acetylcholinesterase inhibitors and a muscarinic receptor agonist in Rhesus monkeys.

Acetylcholinesterase (AChE) inhibitors and positive allosteric nicotinic acetylcholine receptor (nAChR) modulators are potential pharmacotherapies for nicotine dependence. Because some smoking cessation aids (e.g. varenicline) appear to work by mimicking the effects of nicotine, we used drug discrimination to examine whether AChE inhibitors and nAChR allosteric modulators mimic the effects of nicotine. Rhesus monkeys discriminated 1.78 mg/kg of nicotine s.c. under an FR5 schedule of stimulus-shock termination. Nicotine and the AChE inhibitors donepezil and galantamine dose-dependently increased responding on the nicotine-appropriate lever with ED50 values of 0.35, 0.22, and 0.77 mg/kg, respectively. Donepezil (0.56 mg/kg) produced nicotine-like effects for at least 6 h, whereas the duration of action of galantamine (1.78 mg/kg) was less than 3 h. The positive allosteric nAChR modulator PNU-120596 (up to 10 mg/kg) and midazolam (up to 1.0 mg/kg) produced no more than 22% nicotine-lever responding. Oxotremorine, a muscarinic acetylcholine receptor agonist that was used to explore the extent to which muscarinic receptor agonism might contribute to the effects of AChE inhibitors, produced 94% nicotine-lever responding (ED50 value 0.013 mg/kg). The muscarinic antagonist atropine significantly antagonized the effects of both oxotremorine and nicotine; however, the dose of atropine antagonizing oxotremorine was smaller than the dose required to antagonize nicotine. Collectively, these results suggest that AChE inhibitors can mimic the effects of nicotine by indirectly stimulating both nicotinic and muscarinic receptors. Inasmuch as some smoking cessation aids work by exerting nicotine-like effects, the current results are consistent with the potential use of AChE inhibitors as novel smoking cessation aids.

Green tobacco sickness: mecamylamine, varenicline, and nicotine vaccine as clinical research tools and potential therapeutics.

INTRODUCTION: Green tobacco sickness occurs from transdermal absorption of chemicals from freshly harvested, green tobacco leaves. Signs and symptoms include nausea, vomiting, headache, and abdominal cramps. Prevalence has shifted from the United States and Europe to China, India, and Brazil. Worldwide 8 million individuals are afflicted, including women and children. Areas covered: Mecamylamine (Inversine®, Vecamyl®), a nicotinic acetylcholine receptor (nAChR) antagonist, should be tested as a remedy for green tobacco sickness. Mecamylamine is approved as an oral tablet for the treatment of hypertension, is safe, and is off-patent. Mecamylamine attenuates many of the effects of nicotine and tobacco including seizures, thereby supporting its use as an effective pharmacotherapy for tobacco dependence. Varenicline (Chantix®) and cytisine (Tabex®) are low efficacy (i.e. intrinsic activity) nAChR agonists, are used as smoking cessation aids, and are viable options to test as remedies against green tobacco sickness. Nicotine immunization strategies may provide further options for future testing. Expert commentary: Efforts to demonstrate reversal and/or prevention of green tobacco sickness by mecamylamine will underscore the importance of nicotine in this illness and highlight a new medication for effective treatment of tobacco poisoning.

Differential cross-tolerance to the effects of nicotinic acetylcholine receptor drugs in C57BL/6J mice following chronic varenicline.

Varenicline is a smoking cessation pharmacotherapy with a presumed mechanism of action of partial efficacy at the α4ß2 nicotinic acetylcholine receptor (nAChR); however, the extent to which daily varenicline use leads to changes in nAChR sensitivity is unclear. This study examined the consequences of daily varenicline treatment on disruptions in operant responding (i.e. rate-decreasing effects) and hypothermia induced by administration of nicotine, epibatidine, cytisine, and cocaine in C57BL/6J mice. Furthermore, mecamylamine was used to assess the involvement of nAChRs in the effects of varenicline. Mice were trained under a fixed ratio 20 of milk reinforcement, and rectal temperatures were measured after 30 min following drug-administration. Varenicline, nicotine, epibatidine, and cytisine produced dose-dependent decreases in response rate and rectal temperature. Chronic varenicline (30 mg/kg) engendered tolerance to varenicline, but more cross-tolerance to nicotine, for both disruptions in operant responding and hypothermia. Cross-tolerance only developed to the hypothermic effects of epibatidine, and no cross-tolerance developed to any effects of cytisine and cocaine. In varenicline-tolerant mice, mecamylamine did not antagonize the effects of varenicline. The varying magnitudes of tolerance and cross-tolerance among effects and drugs are indicative of a nonuniform nAChR pharmacology in vivo.

Rapid nicotine tolerance and cross-tolerance to varenicline in rhesus monkeys: Drug discrimination.

Acute tolerance to effects of nicotine plays an important role in nicotine dependence, but the mechanism underlying these effects is unclear. Drug discrimination was used in the current study to examine the impact of nicotine pretreatment on sensitivity to the discriminative stimulus effects of nicotine and the FDA-approved smoking cessation pharmacotherapy varenicline. Rhesus monkeys (n = 4) discriminated 0.032 mg/kg nicotine base iv from saline under an FR5 schedule of stimulus-shock termination. Both nicotine and varenicline increased drug-appropriate responding; ED50 values (95% confidence limits) were 0.0087 [0.0025, 0.030] and 0.028 [0.0096, 0.082] mg/kg, respectively. Additional pretreatment injections of the training dose of nicotine (0.032 mg/kg, iv) produced tolerance to its discriminative stimulus effects and the magnitude of this effect was related to the number of pretreatment injections administered. Two pretreatment injections of the training dose of nicotine (0.032 mg/kg, iv) produced a 5.4-fold rightward shift in the nicotine dose-response function and a sevenfold rightward shift in the varenicline dose-response function. The duration of tolerance under these conditions was less than 60 min. These results demonstrate that tolerance to the discriminative stimulus effects of nicotine can be produced by acute nicotine exposure. Acute cross-tolerance from nicotine to varenicline is consistent with similar actions at nAChRs, and suggests that conditions resulting in acute nicotine tolerance could impact sensitivity to other nAChR agonists. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

The contribution of α4ß2 and non-α4ß2 nicotinic acetylcholine receptors to the discriminative stimulus effects of nicotine and varenicline in mice.

RATIONALE: The extent to which non-α4ß2 versus α4ß2* nAChRs contribute to the behavioral effects of varenicline and other nAChR agonists is unclear. OBJECTIVES: The purpose of this study was to characterize the discriminative stimulus effects of varenicline and nicotine using various nAChR agonists and antagonists to elucidate possible non-α4ß2 nAChR mechanisms. METHODS: Separate groups of male C57BL/6J mice were trained to discriminate varenicline (3.2 mg/kg) or nicotine (1 mg/kg). Test drugs included mecamylamine; the nAChR agonists epibatidine, nicotine, cytisine, varenicline, and RTI-102; the ß2-containing nAChR antagonist dihydro-ß-erythroidine (DHßE); the α7 nAChR agonist PNU-282987; the α7 antagonist methyllycaconitine (MLA); the α3ß4 antagonist 18-methoxycoronaridine (18-MC); and the non-nAChR drugs midazolam and cocaine. RESULTS: In nicotine-trained mice, maximum nicotine-appropriate responding was 95% nicotine, 94% epibatidine, 63% varenicline, 58% cytisine, and less than 50% for RTI-102, PNU-282987, midazolam, and cocaine. In varenicline-trained mice, maximum varenicline-appropriate responding was 90% varenicline, 86% epibatidine, 74% cytisine, 80% RTI-102, 50% cocaine, and 50% or less for nicotine, PNU-282987, and midazolam. Drugs were studied to doses that abolished operant responding. Mecamylamine antagonized the discriminative stimulus effects, but not the rate-decreasing effects, of nicotine and varenicline. DHßE antagonized the discriminative stimulus and rate-decreasing effects of nicotine but not varenicline in either the nicotine or varenicline discrimination assays. The discriminative stimulus, but not the rate-decreasing, effects of epibatidine were antagonized by DHßE regardless of the training drug. CONCLUSIONS: These results suggest that α4ß2* nAChRs differentially mediate the discriminative stimulus effects of nicotine and varenicline, and suggest that varenicline has substantial non-α4ß2 nAChR activity.

The discriminative stimulus effects of i.v. nicotine in rhesus monkeys: Pharmacokinetics and apparent pA2 analysis with dihydro-ß-erythroidine.

Quantitative analysis of antagonism is infrequently used to identify nAChRs mediating behavioral effects. Here, nicotine (0.032 mg/kg i.v.) was established as a discriminative stimulus in rhesus monkeys responding under a fixed ratio 5 schedule; pharmacokinetics and underlying nAChR mechanism(s) were examined. When measured up to 4 h in venous blood, the training dose resulted in the following mean pharmacokinetic parameters: nicotine Cmax = 71.7 ng/ml, t1/2 = 116 min, and clearance = 6.25 ml/min/kg; cotinine Cmax = 191 ng/ml; and 3OH-cotinine Cmax = 63 ng/ml. The ED50 value of nicotine to produce discriminative stimulus effects was 0.013 mg/kg. Epibatidine and varenicline increased drug-lever responding to 97% and 95%, respectively (ED50 values = 0.00015 and 0.031 mg/kg, respectively), whereas cocaine, midazolam, and morphine produced no more than 28% drug-appropriate responding. Mecamylamine and dihydro-ß-erythroidine (DHßE) dose-dependently attenuated the discriminative stimulus effects of the nicotine training dose, whereas methyllycaconitine (MLA) did not. DHßE (0.1 and 0.32) produced rightward shifts of the nicotine and varenicline dose-response functions; Schild plots fitted through individual data resulted in slopes that were not different from unity; the apparent pA2 calculated for DHßE did not significantly differ in the presence of nicotine (6.58) or varenicline (6.45). Compared to human cigarette smoking, nicotine blood levels after 0.032 mg/kg nicotine i.v. took a similar time to reach maximal concentration, levels at Cmax were similar to smoking 2-3 cigarettes, while average nicotine levels were comparable to smoking 5-6 cigarettes. Apparent pA2 analysis with DHßE under these conditions is consistent with nicotine and varenicline acting through the same nAChRs to produce discriminative stimulus effects.

Attenuated nicotine-like effects of varenicline but not other nicotinic ACh receptor agonists in monkeys receiving nicotine daily.

BACKGROUND AND PURPOSE: Chronic treatment can differentially impact the effects of pharmacologically related drugs that differ in receptor selectivity and efficacy. EXPERIMENTAL APPROACH: The impact of daily nicotine treatment on the effects of nicotinic ACh receptor (nAChR) agonists was examined in two groups of rhesus monkeys discriminating nicotine (1.78 mg·kg-1 base weight) from saline. One group received additional nicotine treatment post-session (1.78 mg·kg-1 administered five times daily, each dose 2 h apart; i.e. Daily group), and the second group did not (Intermittent group). KEY RESULTS: Daily repeated nicotine treatment produced a time-related increase in saliva cotinine. There was no significant difference in the ED50 values of the nicotine discriminative stimulus between the Daily and Intermittent group. Mecamylamine antagonized the effects of nicotine, whereas dihydro-ß-erythroidine did not. Midazolam produced 0% nicotine-lever responding. The nAChR agonists epibatidine, RTI-36, cytisine and varenicline produced >96% nicotine-lever responding in the Intermittent group. The respective maximum effects in the Daily group were 100, 72, 59 and 28%, which shows that the ability of varenicline to produce nicotine-like responding was selectively decreased in the Daily as compared with the Intermittent group. When combined with nicotine, both varenicline and cytisine increased the potency of nicotine to produce discriminative stimulus effects. CONCLUSION AND IMPLICATIONS: Nicotine treatment has a greater impact on the sensitivity to the effects of varenicline as compared with some other nAChR agonists. Collectively, these results strongly suggest that varenicline differs from nicotine in its selectivity for multiple nAChR subtypes.

Effects of nicotine in combination with drugs described as positive allosteric nicotinic acetylcholine receptor modulators in vitro: discriminative stimulus and hypothermic effects in mice.

Some drugs that are positive allosteric nAChR modulators in vitro, desformylflustrabromine (dFBr), PNU-120596 and LY 2087101, have not been fully characterized in vivo. These drugs were examined for their capacity to share or modify the hypothermic and discriminative stimulus effects of nicotine (1mg/kg s.c.) in male C57Bl/6J mice. Nicotine, dFBr, and PNU-120596 produced significant hypothermia, whereas LY 2087101 (up to 100mg/kg) did not. Nicotine dose-dependently increased nicotine-appropriate responding and decreased response rate; the respective ED50 values were 0.56mg/kg and 0.91mg/kg. The modulators produced no more than 38% nicotine-appropriate responding up to doses that disrupted operant responding. Rank order potency was the same for hypothermia and rate-decreasing effects: nicotine>dFBr>PNU-120596=LY 2087101. Mecamylamine and the α4ß2 nAChR antagonist dihydro-ß-erythroidine, but not the α7 antagonist methyllycaconitine, antagonized the hypothermic effects of nicotine. In contrast, mecamylamine did not antagonize the hypothermic effects of the modulators. The combined discriminative stimulus effects of DFBr and nicotine were synergistic, whereas the combined hypothermic effects of nicotine with either dFBr or PNU-120596 were infra-additive. PNU-120596 did not modify the nicotine discriminative stimulus, and LY 2087101 did not significantly modify either effect of nicotine. Positive modulation of nicotine at nAChRs by PNU-120596 and LY 2087101 in vitro does not appear to confer enhancement of the nAChR-mediated hypothermic or discriminative stimulus effects of nicotine. However, dFBr appears to be a positive allosteric modulator of some behavioral effects of nicotine at doses of dFBr smaller than the doses producing unwanted effects (e.g. hypothermia) through non-nAChR mechanisms.

Differential antagonism and tolerance/cross-tolerance among nicotinic acetylcholine receptor agonists: scheduled-controlled responding and hypothermia in C57BL/6J mice.

The tobacco-dependence pharmacotherapies varenicline and cytisine act as partial α4ß2 nAChR agonists. However, the extent to which α4ß2 nicotinic acetylcholine receptors (nAChRs) mediate their in-vivo effects remains unclear. Nicotine, varenicline, cytisine, and epibatidine were studied in male C57BL/6J mice for their effects on rates of fixed ratio responding and rectal temperature alone and in combination with the nonselective nAChR antagonist mecamylamine and the α4ß2 nAChR antagonist dihydro-ß-erythroidine. The effects of nicotine, varenicline, cytisine, epibatidine, and cocaine were assessed before and during chronic nicotine treatment. The rate-decreasing and hypothermic effects of nicotine, varenicline, cytisine, and epibatidine were antagonized by mecamylamine (1 mg/kg), but only the effects of nicotine and epibatidine were antagonized by dihydro-ß-erythroidine (3.2 mg/kg). Chronic nicotine produced 4.7 and 5.1-fold rightward shifts in the nicotine dose-effect functions to decrease response rate and rectal temperature, respectively. Nicotine treatment decreased the potency of epibatidine to decrease response rate and rectal temperature 2.2 and 2.9-fold, respectively, and shifted the varenicline dose-effect functions 2.0 and 1.7-fold rightward, respectively. Cross-tolerance did not develop from nicotine to cytisine. These results suggest that the in-vivo pharmacology of tobacco cessation aids cannot be attributed to a single nAChR subtype; instead, multiple receptor subtypes differentially mediate their effects.

The discriminative stimulus effects of mecamylamine in nicotine-treated and untreated rhesus monkeys.

The extent to which chronic nicotine treatment can alter the effects of the nicotinic acetylcholine receptor antagonist mecamylamine, and whether those effects can be attenuated by nicotine have not been clearly established in the literature. Here, the discriminative stimulus effects of mecamylamine were compared between one group of rhesus monkeys receiving a continuous infusion of nicotine base (5.6 mg/kg/day subcutaneously) and another group of monkeys not receiving nicotine treatment. Both groups responded under a fixed ratio 5 schedule of stimulus-shock termination. Stimulus control was obtained at doses of 1.78 mg/kg mecamylamine in monkeys receiving continuous nicotine and 5.6 mg/kg mecamylamine in monkeys not receiving continuous nicotine treatment. Nicotine did not attenuate the discriminative stimulus effects of mecamylamine in either group. Discontinuation of continuous nicotine produced responding on the mecamylamine lever within 24 h in some but not all monkeys. This may indicate a qualitative difference in the discriminative stimulus effects of mecamylamine between groups, perhaps reflecting antagonism of nicotine and nicotine withdrawal in monkeys receiving continuous nicotine. The failure of nicotine to reverse the effects of mecamylamine is consistent with a noncompetitive interaction at nicotinic acetylcholine receptors and indicates that mecamylamine-induced withdrawal cannot be readily modified by nicotine.

Discriminative stimulus and hypothermic effects of some derivatives of the nAChR agonist epibatidine in mice.

RATIONALE: Receptor mechanisms underlying the in vivo effects of nicotinic acetylcholine receptor (nAChR) drugs need to be determined to better understand possible differences in therapeutic potential. OBJECTIVE: This study compared the effects of agonists that are reported either to differ in intrinsic activity (i.e., efficacy) at α4ß2 nAChR in vitro or to have in vivo effects consistent with differences in efficacy. The drugs included nicotine, varenicline, cytisine, epibatidine, and three novel epibatidine derivatives: 2'-fluoro-3'-(4-nitrophenyl)deschloroepibatidine (RTI-7527-102), 2'-fluorodeschloroepibatidine (RTI-7527-36), and 3'-(3″-dimethylaminophenyl)-epibatidine (RTI-7527-76). METHODS: Mice discriminated nicotine base (1 mg/kg base) from saline; other mice were used to measure rectal temperature. RESULTS: In the nicotine discrimination assay, the maximum percentage of nicotine-appropriate responding varied: 92 % for nicotine, 84 % for epibatidine, 77 % for RTI-7527-36, and 71 % for varenicline and significantly less for RTI-7527-76 (58 %), RTI-7527-102 (46 %), and cytisine (33 %). Each drug markedly decreased rectal temperature by as much as 12 ºC. The rank-order potency in the discrimination and hypothermia assays was epibatidine > RTI-7527-36 > nicotine > RTI-7527-102 > varenicline = cytisine = RTI-7527-76. The nAChR antagonist mecamylamine (3.2 mg/kg) antagonized the discriminative stimulus effects of epibatidine and RTI-7527-102, as well as the hypothermic effects of every drug except cytisine. The ß2-subunit selective nAChR antagonist dihydro-ß-erythroidine (DHßE; up to 10 mg/kg) antagonized hypothermic effects but less effectively so than mecamylamine. CONCLUSIONS: The marked hypothermic effects of all drugs except cytisine are due in part to agonism at nAChR containing ß2-subunits. Differential substitution for the nicotine discriminative stimulus is consistent with differences in α4ß2 nAChR efficacy; however, collectively the current results suggest that multiple nAChR receptor subtypes mediate the effects of the agonists.